The Thr505 and Ser557 residues of the AGT1-encoded alpha-glucoside transporter are critical for maltotriose transport in Saccharomyces cerevisiae

Aims:  The main objective of this study was to identify amino acid residues in the AGT1‐encoded α‐glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae. Methods and Results:  The sequences of two AGT1‐encoded α‐glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1p_WH310 and Agt1p_WH314) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1‐encoded α‐glucoside transporters. A 23‐amino‐acid C‐terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport. Conclusions:  The amino acids Thr⁵⁰⁵ and Ser⁵⁵⁷, which are respectively located in the transmembrane (TM) segment TM¹¹ and on the intracellular segment after TM¹² of the AGT1‐encoded α‐glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae. Significance and Impact of the Study:  Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch‐efficient fermentation systems, and offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whisky industries.     

Life Expectancy in a Large Cohort of Type 2 Diabetes Patients Treated in Primary Care (ZODIAC-10)

Background

Most longitudinal studies showed increased relative mortality in individuals with type 2 diabetes mellitus until now. As a result of major changes in treatment regimes over the past years, with more stringent goals for metabolic control and cardiovascular risk management, improvement of life expectancy should be expected. In our study, we aimed to assess present-day life expectancy of type 2 diabetes patients in an ongoing cohort study.

Methodology and Principal Findings

We included 973 primary care type 2 diabetes patients in a prospective cohort study, who were all participating in a shared care project in The Netherlands. Vital status was assessed from May 2001 till May 2007. Main outcome measurement was life expectancy assessed by transforming actual survival time to standardised survival time allowing adjustment for the baseline mortality rate of the general population. At baseline, mean age was 66 years, mean HbA_1c 7.0%. During a median follow-up of 5.4 years, 165 patients died (78 from cardiovascular causes), and 17 patients were lost to follow-up. There were no differences in life expectancy in subjects with type 2 diabetes compared to life expectancy in the general population. In multivariate Cox regression analyses, concentrating on the endpoints ‘all-cause’ and cardiovascular mortality, a history of cardiovascular disease: hazard ratio (HR) 1.71 (95% confidence interval (CI) 1.23–2.37), and HR 2.59 (95% CI 1.56–4.28); and albuminuria: HR 1.72 (95% CI 1.26–2.35), and HR 1.83 (95% CI 1.17–2.89), respectively, were significant predictors, whereas smoking, HbA_1c, systolic blood pressure and diabetes duration were not.

Conclusions

This study shows a normal life expectancy in a cohort of subjects with type 2 diabetes patients in primary care when compared to the general population. A history of cardiovascular disease and albuminuria, however, increased the risk of a reduction of life expectancy. These results show that, in a shared care environment, a normal life expectancy is achievable in type 2 diabetes patients.     

Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.     

Cycloheximide resistance in the Lipomycetaceae revisited

Cycloheximide (CYH) is a heterocyclic, glutarimide antibiotic that is a potent inhibitor of protein biosynthesis in most eukaryotes. This study demonstrated that yeasts from all species of the Lipomycetaceae, with the exception of Dipodascopsis spp., can grow in the presence of up to 5 g.L(-1) CYH -- a concentration that is five times higher than the accepted "highest" concentration of 1 g.L(-1) used in physiological tests for yeast identification. Lipomycetaceous yeasts are known to utilize heterocyclic nitrogen-containing compounds such as thymine as sole nitrogen source. CYH contains a glutarimide ring, which is chemically similar to thymine. We investigated the possibility that CYH resistance in the Lipomycetaceae might be due to an ability to degrade CYH and use it as the sole nitrogen source. However, we were unable to demonstrate significant growth on CYH as sole nitrogen source. When thymine was used as positive control, we could demonstrate its utilization as sole nitrogen source.     

Insulin Resistance and Risk of Incident Cardiovascular Events in Adults without Diabetes: Meta-Analysis

Background

Glucose, insulin and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) are markers of insulin resistance. The objective of this study is to compare fasting glucose, fasting insulin concentrations and HOMA-IR in strength of association with incident cardiovascular disease.

Methods

We searched the PubMed, MEDLINE, EMBASE, Web of Science, ScienceDirect and Cochrane Library databases from inception to March, 2011, and screened reference lists. Cohort studies or nested case-control studies that investigated the association between fasting glucose, fasting insulin or HOMA-IR and incident cardiovascular disease, were eligible. Two investigators independently performed the article selection, data extraction and risk of bias assessment. Cardiovascular endpoints were coronary heart disease (CHD), stroke or combined cardiovascular disease. We used fixed and random-effect meta-analyses to calculate the pooled relative risk for CHD, stroke and combined cardiovascular disease, comparing high to low concentrations of glucose, insulin or HOMA-IR. Study heterogeneity was calculated with the I² statistic. To enable a comparison between cardiovascular disease risks for glucose, insulin and HOMA-IR, we calculated pooled relative risks per increase of one standard deviation.

Results

We included 65 studies (involving 516,325 participants) in this meta-analysis. In a random-effect meta-analysis the pooled relative risk of CHD (95% CI; I²) comparing high to low concentrations was 1.52 (1.31, 1.76; 62.4%) for glucose, 1.12 (0.92, 1.37; 41.0%) for insulin and 1.64 (1.35, 2.00; 0%) for HOMA-IR. The pooled relative risk of CHD per one standard deviation increase was 1.21 (1.13, 1.30; 64.9%) for glucose, 1.04 (0.96, 1.12; 43.0%) for insulin and 1.46 (1.26, 1.69; 0.0%) for HOMA-IR.

Conclusions

The relative risk of cardiovascular disease was higher for an increase of one standard deviation in HOMA-IR compared to an increase of one standard deviation in fasting glucose or fasting insulin concentration. It may be useful to add HOMA-IR to a cardiovascular risk prediction model.     

Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon

CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85?μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6–0.8?μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5–1.0?μmol P450 g _DCW ^?1 , for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7?g octanol L _BRM ^?1 was obtained within 24?h (0.34?g L _BRM ^?1 ?h^?1) with IPTG-induced cells containing only 0.20?μmol P450 g _DCW ^?1 , when glucose (22?g L _BRM ^?1 ) was added for cofactor regeneration.     

Endoplasmic reticulum-quality control chaperones facilitate the biogenesis of Cf receptor-like proteins involved in pathogen resistance of tomato

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.     

Differential Anti-Glycan Antibody Responses in Schistosoma mansoni-Infected Children and Adults Studied by Shotgun Glycan Microarray

Background

Schistosomiasis (bilharzia) is a chronic and potentially deadly parasitic disease that affects millions of people in (sub)tropical areas. An important partial immunity to Schistosoma infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during Schistosoma infection are directed against the numerous glycans expressed by Schistosoma. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown.

Methodology/Principal Findings

The binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of S. mansoni and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection.

Conclusions/Significance

Using the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in S. mansoni infections. These findings are significant for further exploration of Schistosoma glycan antigens in relation to immunity.